Cloning and Functional Study of the AbWRKY1 Gene from Atropa belladonna
PAN Hang, MU De-Hui, YANG Jia, TAN Ai-Juan, MA Bing-Nan, XU Guo-Ju, YANG Li-Hang, QIANG Wei*
College of Life Sciences/Research Institute of Agricultural Biological Engineering, Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China
Abstract：Atropa belladonna is a commercial medicinal plant of tropane alkaloids (TAs) listed in the 《Chinese Pharmacopoeia》, and WRKY transcription factors may regulate the biosynthesis of TAs. In this study, tissue expression profile, phylogenetic tree and inducible expression analyses of the WRKY transcription factor family in A. belladonna were performed to screen out WRKY gene that positively regulated TAs biosynthesis. AbWRKY1 was identified and its function was verified at the biochemical and molecular genetic levels. AbWRKY1 and TAs synthetic pathway genes shared the same tissue and inducible expression characteristics that they were specifically expressed in the adventitious roots and were induced by the calcium channel inhibitor verapamil. AbWRKY1 clustered together with WRKYs from other species that had functions in regulating secondary metabolism. AbWRKY1 protein contained 326 amino acid residues, and localized in nuclear. Yeast one-hybrid result showed that AbWRKY1 could interact with the W-box rich region of hyoscyamine 6β-hydroxylase (H6H) gene promoter. Knockdown of AbWRKY1 expression by virus-induced gene silencing (VIGS) in A. belladonna plants reduced scopolamine and hyoscyamine levels by 44.91% and 79.33%, respectively. AbWRKY1 overexpression raised the amount of scopolamine, anisodamine, and hyoscyamine in A. belladonna's hairy roots, while the expression of the 4 TAs synthesis pathway genes, namely putrescine N-methyltransferase (PMT), tropinone reductaseⅠ(TRⅠ), hyoscyamine dehydrogenase (HDH) and H6H, were increased concomitantly. This study provides a theoretical basis for the elucidation of the regulatory mechanism of TAs biosynthesis and the molecular breeding of A. belladonna.
潘航, 穆德会, 杨嘉, 谭艾娟, 马炳南, 许国菊, 杨立航, 强玮. 颠茄AbWRKY1基因的克隆与功能研究[J]. 农业生物技术学报, 2025, 33(2): 316-329.
PAN Hang, MU De-Hui, YANG Jia, TAN Ai-Juan, MA Bing-Nan, XU Guo-Ju, YANG Li-Hang, QIANG Wei. Cloning and Functional Study of the AbWRKY1 Gene from Atropa belladonna. 农业生物技术学报, 2025, 33(2): 316-329.
[1] 敖雯雯, 谭艾娟, 马炳南, 等. 2023. rolB基因对颠茄表型发育和托品烷生物碱合成的影响[J]. 药学学报, 58(6): 1705-1712. (Ao W W, Tan A J, Ma B N, et al.2023. Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna[J]. Acta Pharmaceutica Sinica, 58(6): 1705-1712.) [2] 付维, 郑涛, 姜育松, 等. 2020. 颠茄AbCYP80F1基因的克隆与功能分析[J]. 中草药, 51: 4291-4299. (Fu W, Zheng T, Jiang Y S, et al.2020. Cloning and functional analysis of AbCYP80F1 gene of Atropa belladonna[J]. Chinese Traditional and Herbal Drugs, 51: 4291-4299.) [3] 路星星, 谭艾娟, 周浩, 等. 2022. rolC基因对颠茄托品烷生物碱合成的影响[J]. 药学学报, 57(2): 533-540. (Lu X X, Tan A J, Zhou H, et al.2022. Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna.[J]. Acta Pharmaceutica Sinica, 57(2): 533-540.) [4] 李笑, 强玮, 邱飞, 等. 2017. 天仙子中参与托品烷生物碱生物合成的ArAT基因的克隆及功能鉴定[J]. 药学学报, 52(1): 172-179. (Li X, Qiang W, Qiu F, et al.2017. Cloning and characterization of an aromatic amino acid aminotransferase (ArAT) gene involved in tropane alkaloid biosynthesis from Hyoscyamus niger[J]. Acta Pharmaceutica Sinica, 52(1): 172-179.) [5] 卢衍, 王细荣, 陈敏, 等. 2012. 托品烷类生物碱生物合成分子生物学与代谢工程[J]. 中草药,43(05):1018-1023. (Lu Y, Wang X R, Chen M, et al.2012. Molecular biology and metabolic engineering of tropane alkaloids biosynthesis[J]. Chinese Traditional and Herbal Drugs, 43(5): 1018-1023.) [6] 穆德会, 刘艳红, 陈漂漂, 等. 2023. 超表达HnCYP82M3和DsTRI基因对颠茄托品烷生物碱合成的影响[J]. 药学学报, 1-18. (Mu D H, Liu Y H, Chen P P, et al.2023. Enhancement of tropane alkaloids biosynthesis in Atropa belladonna hariy root by overexpression of HnCYP82M3 and DsTRI genes[J]. Acta Pharmaceutica Sinica , 1-18.) [7] 强玮, 王亚雄, 张巧卓, 等. 2014. 颠茄托品烷生物碱合成途径基因表达分析与生物碱积累研究[J]. 中国中药杂志, 39(1): 52-58. (Qiang W, Wang YX, Zhang QZ, et al.2014. Expression pattern of genes involved in tropane alkaloids biosynthesis and tropane alkaloids accumulation in Atropa belladonna[J]. China Journal of Chinese Materia Medica, 39(1): 52-58.) [8] 武志刚,武舒佳,王迎春,等. 2018. 植物中钙依赖蛋白激酶(CDPK)的研究进展[J]. 草业学报, 27(1): 204-214. (Wu Z G, Wu S J, Wang Y C, et al.2018. Advances in studies of calcium-dependent protein kinase (CDPK) in plants[J]. Acta Prataculturae Sinica, 27(1): 204-214.) [9] 杨卫星, 黑刚刚, 李琳琳, 等. 2014. 钙对昼间亚高温胁迫下半夏质量和产量的影响[J]. 西南大学学报(自然科学版), 36(8): 20-26. (Yang W X, Hei G G, Li L L, et al.2014. Effect of calcium on the yield and quality of Pinellia ternate under dayingtime sub-high temperature stress[J]. Journal of Southwest University (Natural Science Edition), 36(8): 20-26.) [10] 周浩, 路星星, 敖雯雯, 等. 2022. 钩藤STR基因及其启动子的克隆与分析[J]. 药学学报, 57(5): 1526-1536. (Zhou H, Lu X X, Ao W W, et al.2022. Cloning and analysis of STR gene and its promoter from Uncaria[J]. Acta Pharmaceutica Sinica, 57(5): 1526-1536.) [11] 郑浩杰, 杭烨, 王晓红, 等2022. 药用植物WRKY转录因子家族研究进展[J]. 植物生理学报, 58(6): 1055-1067. (Zheng H J, Hang Y, Wang X H, et al.2021. Research progress of WRKY transcription factor family in medicinal plants[J]. Plant Physiology Journal, 58(6): 1055-1067.) [12] Cao W, Wang Y, Shi M, et al.2018. Transcription factor SmWRKY1 positively promotes the biosynthesis of tanshinones in Salvia miltiorrhiza[J]. Frontiers in Plant Science, 9: 554. [13] Chen Y, Zhang H, Zhang M, et al.2021. Salicylic acid-responsive factor TcWRKY33 positively regulates taxol biosynthesis in Taxus chinensis in direct and indirect ways[J]. Frontiers in Plant Science, 12:697476. [14] Eulgem T J, Rushton P, Schmelzer E, et al.1999. Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors[J]. The EMBO Journal, 18(17): 4689-4699. [15] Eulgem T, Rushton P J, Robatzek S, et al.2000. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 5(5): 199-206. [16] Gou Y Q, Jing Y M, Song J X, et al.2024. A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna[J].International Journal of Biological Macromolecules, 266(1): 131012. [17] Hao X, Xie C, Ruan Q, et al.2021. The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila[J]. Horticulture Research, 8: 7. [18] Huang J P, Wang Y J, Tian T, et al.2021. Tropane alkaloid biosynthesis: A centennial review[J]. Natural Product Reports, 38(9): 1634-1658. [19] Jiang J, Ma S, Ye N, et al.2017. WRKY transcription factors in plant responses to stresses: WRKY in plant responses to stresses[J]. Journal of Integrative Plant Biology, 59(2): 86-101. [20] Jiang W, Fu X, Pan Q, et al.2016. Overexpression of AaWRKY1 leads to an enhanced content of artemisinin in Artemisia annua[J]. BioMed Research International 2016:1-9. [21] Kai G, Yang S, Luo X, et al.2011. Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots[J]. BMC Biotechnol, 11: 43. [22] Kai G Y, Zhang A, Guo Y, et al.2012. Enhancing the production of tropane alkaloids in transgenic Anisodus acutangulus hairy root cultures by over-expressing tropinone reductaseⅠand hyoscyamine-6β-hydroxylase[J]. Molecular bioSystems, 8(11): 2883-2890. [23] Lange M, Yellina A L, Orashakova S, et al.2013. Virus-induced gene silencing (VIGS) in plants: An overview of target species and the virus-derived vector systems[J]. Methods in Molecular Biology, 975: 1-14. [24] Qiu F, Yan Y, Zeng J, et al.2021. Biochemical and metabolic insights into hyoscyamine dehydrogenase[J]. ACS Catalysis, 11(5): 2912-2924. [25] Rössner C, Lotz D, Becker A.2022. VIGS goes viral: How VIGS transforms our understanding of plant science[J]. Annual Review Plant Biology, 73: 703-728. [26] Rushton P J, Somssich I E, Ringler P, et al.2010. WRKY transcription factors[J]. Trends in Plant Science, 15(5): 247-258. [27] SuttipantaN, Pattanaik S, KulshresthaM, et al.2011. The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J].Plant Physiology, 157(4): 2081-2093. [28] Xia K, Liu X, Zhang Q, et al.2016. Promoting scopolamine biosynthesis in transgenic Atropa belladonna plants with PMT and H6H overexpression under field conditions[J]. Plant Physiology and Biochemistry, 106: 46-53. [29] Xiao Y, Feng J, Li Q, et al.2020. IiWRKY34 positively regulates yield, lignan biosynthesis and stress tolerance in Isatis indigotica[J]. Acta Pharmaceutica Sinica B, 10(12): 2417-2432. [30] Zaidi S S, Vasudevan K, Lentz E M, et al.2020. Virus-induced gene silencing (VIGS) in cassava using geminivirus agroclones[J]. Methods in Molecular Biology (Clifton, N.J.), 2172: 51-64. [31] Zhang Q Z, Liang M J, Liu Y Yet al.2021. Development of homozygous transgenic Atropa belladonna plants with glyphosate resistance and high-yield scopolamine using metabolic engineering[J]. Industrial Crops & Products, 171: 113953. [32] Zhou W, Wang K, Hao X L, et al.2024. A chromosome-level genome assembly of anesthetic drug-producing anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids[J]. Plant Communications, 5(1): 100680.
